A negatively acting bifunctional RNA increases survival motor neuron both in vitro and in vivo - PubMed (original) (raw)
A negatively acting bifunctional RNA increases survival motor neuron both in vitro and in vivo
Alexa Dickson et al. Hum Gene Ther. 2008 Nov.
Abstract
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder and is the leading genetic cause of infant mortality. SMA is caused by the loss of survival motor neuron-1 (SMN1). In humans, a nearly identical copy gene is present called SMN2, but this gene cannot compensate for the loss of SMN1 because of a single silent nucleotide difference in SMN2 exon 7. This single-nucleotide difference attenuates an exonic splice enhancer, resulting in the production of an alternatively spliced isoform lacking exon 7, which is essential for protein function. SMN2, however, is a critical disease modifier and is an outstanding target for therapeutic intervention because all SMA patients retain SMN2 and SMN2 maintains the same coding sequence as SMN1. Therefore, compounds or molecules that increase SMN2 exon 7 inclusion hold great promise for SMA therapeutics. Bifunctional RNAs have been previously used to increase SMN protein levels and derive their name from the presence of two domains: an antisense RNA sequence specific to the target RNA and an untethered RNA segment that serves as a binding platform for splicing factors. This study was designed to develop negatively acting bifunctional RNAs that recruit hnRNPA1 to exon 8 and block the general splicing machinery from the exon 8. By blocking the downstream splice site, this could competitively favor the inclusion of SMN exon 7 and therefore increase full-length SMN production. Here we identify a bifunctional RNA that stimulated full-length SMN expression in a variety of cell-based assays including SMA patient fibroblasts. Importantly, this molecule was also able to induce SMN expression in a previously described mouse model of SMA and demonstrates a novel therapeutic approach for SMA as well as a variety of diseases caused by a defect in splicing.
Figures
FIG. 1.
Schematic of bifunctional RNAs. (A) Illustration of plasmid-based bifunctional RNA targeting hnRNPA1 to SMN exon 8. (B) Illustration of 2′-_O_-methyl-based bifunctional RNAs. Exon8-hnRNPA1 RNA is identical to the plasmid-based bifunctional RNA. G2 is based on a previously published antisense RNA shown to increase splicing to exon 7, and is the identical antisense to exon8-hnRNPA1. hnRNPA1-act is only the SELEX-determined hnRNPA1 recruitment domain without a targeting antisense. D2-2 hnRNPA1 contains an antisense domain that is shifted 5′ into the exon, relative to exon8-hnRNPA1, and also contains tandem repeats of the SELEX-determined hnRNPA1 recruitment domain. D2-2 is a previously published antisense, but was unable to increase splicing to exon 7.
FIG. 2.
Cotransfection of _SMN2_-luciferase reporter with exon8-hnRNPA1 bifunctional RNA increases luminescence. HeLa cells were transfected with a cassette expressing _SMN2_-luciferase and exon8-hnRNPA1 bifunctional-expressing plasmid. These cells showed a significant increase in luminescence, showing an increase in splicing to exon 7 (p < 0.08 × 10−8).
FIG. 3.
Transfection of exon8-hnRNPA1 bifunctional RNA increases gem numbers in fibroblasts from patients with severe disease. Exon8-hnRNPA1 bifunctional RNA-expressing plasmid was transfected into GM03813 cells (type I patient fibroblast cells) and harvested after 48 hr. Transfected cells were identified by GFP expression and SMN protein was analyzed with a previously published SMN antibody.
FIG. 4.
(A and B) Transfection of 2′-_O_-methyl exon8-hnRNPA1 bifunctional RNA increases gem numbers in fibroblasts from patients with severe disease. 2′-_O_-Methyl bifunctional RNAs were transfected into GM03813 cells (type I patient fibroblast cells) and SMN localization was observed with an SMN-specific antibody 48 hr posttransfection. Gem distribution showed that the majority of transfected cells had one or two gems per nucleus. Exon8-hnRNPA1 transfection significantly increased gem numbers (p < 0.006).
FIG. 5.
Transfection of 2′-_O_-methyl exon8-hnRNPA1 bifunctional RNA increases total SMN protein in fibroblasts from a patient with severe disease. 2′-_O_-Methyl bifunctional RNAs were transfected into GM03813 cells (type I patient fibroblast cells) and total SMN protein was analyzed by Western blotting 48 hr posttransfection.
FIG. 6.
Injection of 2′-_O_-methyl exon8-hnRNPA1 bifunctional RNA increases total SMN protein in the SMA mouse model. (A) Intrafacial vein injection of 2′-_O_-methyl exon8-hnRNPA1 bifunctional RNA increases SMN protein in liver and kidney after 48 hr. (B) Intraventricular injection of 2′-_O_-methyl exon8-hnRNPA1 bifunctional RNA increases SMN protein in brain after 24 hr. Equivalent levels of protein were quantitated and confirmed by staining with actin (data not shown).
References
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